a) Field of the Invention
The present invention relates to methods for the treatment of arrhythmias by inhibition of a multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase), pharmaceutical compositions useful in such treatments, and methods for identifying new agents useful for such treatments.
b) Description of Related Art
Arrhythmias are a leading cause of cardiac-related death in the United States. Prolongation of the cardiac action potential is an important predisposing condition for these arrhythmias. Many antiarrhythmic drugs directly prolong the action potential duration and so may further contribute to these arrhythmias (i.e. the proarrhythmic effects of antiarrhythmic drugs). Despite their cost, implantable cardiac defibrillators (ICDs) have become the treatment of choice for arrhythmias. In order to prevent painful shocks xcx9c50% of patients with ICDs require additional treatment with antiarrhythmic drugs. Thus there is an important need to develop better antiarrhythmic drug therapies.
Early afterdepolarizations (EADs) are depolarizing oscillations in the action potential (AP) that occur during repolarization. One cause of EADs is inward L-type Ca2+ current (ICa). ICa is present at cell membrane potentials (Vm) within the window of ICa steady state activation and inactivation overlap; such as occur during action potential repolarization. Prolongation of action potential repolarization may increase the time that the Vm is in the window current range for ICa and thus the likelihood of EADs. EADs are important because they are one probable cause of lethal arrhythmias associated with long QT intervals including torsade de pointes. A long QT interval reflects prolonged action potential repolarization in ventricular myocardium and is due to a wide variety of conditions including bradycardia and hypokalemia. One important cause of long QT intervals are antiaffhythmic drugs and the ventricular proarrhythmic effects of many antiarrhythmic agents are due to QT interval prolongation.
Intracellular Ca2+ increases simultaneously with EADs in isolated ventricular myocytes (De Ferrari et al. (1995) Circ 91:2510-2515). Elevation of intracellular Ca2+ ([Ca2+]i) has complex effects on ICa including indirect enhancement through a multifunctional Ca2+/calmodulin-dependent protein kinase II pathway (Anderson et al. (1994) Circ Res 75:854-861) and direct inactivation. The net effect of elevated [Ca2+]i in rabbit ventricular myocytes following flash photolysis of the photolabile Ca2+ chelator Nitrxe2x88x925 is 40-50% augmentation of peak ICa that is mediated by a multifunctional Ca2+/calmodulin-dependent protein kinase II, hereafter referred to as CaM kinase II. CaM kinase II, like other multifunctional Ca2+/calmodulin-dependent protein kinases, is an ubiquitous serine-threonine kinase that is activated when Ca2+ is bound to the Ca2+ binding protein calmodulin. Once activated by Ca2+/calmodulin, CaM kinase II activation may be sustained by intersubunit enzyme autophosphorylation that confers Ca2+-independent activity, allowing for its activity to persist during the long diastolic intervals associated with QT interval prolongation and torsade de pointes. This Ca2+-independent activity is enhanced by long stimulating pulses (De Koninck and Schulman (1998) Science 279:227-230), as occur with prolonged action potential repolarization. It is possible that EADs caused by ICa may be enhanced by increased [Ca2+]i through the CaM kinase II pathway. However, not all EADs are due to ICa, and EADs can occur in conditions adverse to CaM kinase activity such as enhanced [Ca2+]i buffering.
Delayed afterdepolarizations (DADs) are another cause of ventricular arrhythmias associated with intracellular calcium overload. DADs are caused by an inward current that follows completion of action potential repolarization. This inward current is a marker of intracellular calcium overload (Thandroyen et al. (1991) Circ Res 69:810-819). Intracellular calcium overload is a central feature of many ventricular arrhythmias occurring during ischemia (Lee et al. (1988) Circ 78:1047-1059) including ventricular fibrillation.
Inhibition of CaM kinase activity can be used to test for a facilitatory role of CaM kinase in EADs and DADs. There are several methods for blocking CaM kinase activity. Synthetic pseudo-substrate peptide inhibitors of CaM kinase provide a specific approach to CaM kinase inhibition and have been used in a variety of cell types including ventricular myocytes (Braun et al. (1995) J. Physiol 488:37-55). The peptide sequence KKALHRQEAVDCL (SEQ ID NO:1), like other similar peptides, was found to inhibit CaM kinase in a highly specific manner. SEQ ID NO:1 is a much less efficient inhibitor of both protein kinase A (PKA) and protein kinase C (PKC), with an IC50 value of at least 500 xcexcmol/l for each. Myristoylated inhibitory peptides are cell membrane permeant and thus could also be effective when added extracellularly.
A variety of cellxe2x80x94membrane permeant organic CaM kinase and calmodulin inhibitors are available and widely used (Braun et al. (1995) Ann Rev Physiol 57:417-445). A principle disadvantage for these inhibitors as experimental agents is that many of them directly block ICa.
However, direct ICa blockade by the CaM, kinase inhibitor KN-93 has not been previously reported. KN-93 (2-[N-(2-hydroxyethyl)-N-(4-methoxy-benzenesulfonyl)]-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) is a methoxybenzene sulfonamide derivative that competitively inhibits calmodulin binding to CaM kinase with a reported Ki of 0.37 xcexcmol/l. KN-93 has been shown to inhibit CaM kinasexe2x80x94dependent processes in PC12h cells, fibroblasts, and gastric parietal cells. There are four isoform groups of CaM kinase II (xcex1,xcex2,xcex3,xcex4) and the xcex4B and xcex4C isoforms have been identified in myocardium. The catalytic and regulatory domains in CaM kinase are highly conserved in all known CaM kinase isoforms so inhibitors that interact with either of these domains are expected to work in all cell types including cardiac. KN-92 (2-N-(4-methoxybenzenesulfonyl)-amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) is a congener of KN-93 without CaM kinase inhibitory activity and is used as an experimental control. Direct ICa blockade by KN-92 has also not been previously reported, and neither KN-93 nor KN-92 have appreciable effects on other serine threonine kinases such as protein kinase A (PKA) or protein kinase C (PKC).
Two general approaches are currently used to suppress ventricular arrhythmias due to action potential prolongation, in addition to ICD implantation. One is to shorten the action potential using antiarrhythmic agents (e.g. mexilitine, pinacidil) or by increasing the heart rate using artificial pacing or the xcex2-adrenergic agent isoproterenol. The second is to indirectly suppress protein kinase A (PKA), which enhances L-type calcium current and calcium release from the sarcoplasmic reticulum, through left stellate ganglionectomy or with xcex2-adrenergic blocking drugs. Neither of these approaches is broadly applicable for two reasons: 1) action potential duration is governed by a number of different ionic currents and it is not typically known which current is critical in a given patient. Furthermore, specific antiarrhythmic drugs are not available for modification of many of these ionic currents. In addition, not all causes of action potential prolongation respond to pacing by action potential shortening. Isoproterenol also does not always shorten the action potential, can itself be arrhythmogenic, may cause ischemia, and can only be used in an acute setting. 2) At present, suppression of PKA is accomplished indirectly by stellate gangionectomy or by xcex2-adrenergic antagonists. xcex2-adrenergic antagonists are effective in reducing death from arrhythmias, but application is limited by the fact that these agents weaken the force of heart muscle contraction.
Ventricular arrhythmias due to ischemia and intracellular calcium overload are generally treated with revascularization (i.e. coronary artery angioplasty or coronary artery bypass surgery), xcex2-adrenergic antagonists, and class III antiarrhythmic medications (e.g. sotalol and amiodarone), in addition to ICDs. Unfortunately revascularization is often incomplete and the recurrence rate of ventricular fibrillation is significant. Class III antiarrhythmic agents may be proarrhythmic by causing excessive action potential prolongation or be associated with usexe2x80x94limiting toxicity (e.g. amiodarone). Beta-adrenergic antagonist use is also limited as previously discussed (above).
Atrial fibrillation is associated with significant morbidity and mortality from stroke and heart failure. Atrial fibrillation can be caused by DADs. Maintenance of atrial fibrillation is favored by intracellular calcium dependent processes. (Tieleman et al. (1997) Circ 95:1945-1953) Present conventional therapies center around anticoagulation (for prevention of stroke), ventricular rate control, and antiarrhythmic agents. Currently available antiarrhythmic agents only succeed in maintaining sinus rhythm in xcx9c50% of patients/year. Recent experimental therapies include artificial pacing systems and atrial ICDs.
CaM kinase inhibition may be superior to previous antiarrhythmic strategies because CaM kinase has characteristics that may allow it to function as a proarrhythmic positive feedback effector for EADs and DADs, and thus play a more central role in EAD and DAD induction than other effectors, such as PKA. L-type calcium current increases intracellular calcium directly by transmembrane calcium flux and indirectly through calcium-induced release of calcium from the sarcoplasmic reticulum. Increased intracellular calcium results in enhanced CaM kinase activity to further favor EADs during action potential prolongation. In contrast, PKA activity is not enhanced by increased intracellular calcium. Direct CaM kinase inhibitors are available (above) and CaM kinase inhibition does not reduce the strength of contraction in isolated hearts, suggesting it may be applicable in patients who do not tolerate xcex2-adrenergic antagonists. Recent experiments using genetically engineered mice lacking neuronal CaM kinase isoforms suggest that systemic CaM kinase inhibition will not result in intolerable side effects. It is likely that redundant CaM kinase isoforms or other signal transduction mechanisms partially compensate for the inactivity of one CaM kinase subtype, so the actual effects of knockout or other inhibition strategies are less deleterious than anticipated. Thus, CaM kinase inhibition may be highly beneficial as a treatment for arrhythmias related to excessive action potential prolongation and EADs and for arrhythmias related to intracellular calcium overload such as atrial and ventricular fibrillation.
Action potential prolongation favors increased L-type calcium current, and this current is the likely proximate cause of the arrhythmias, but because calcium is essential for cardiac muscle function, direct blockade of L-type calcium current is not a viable antiarrhythmic strategy. L-type Ca2+ current inhibitors have not been found to be highly effective antiarrhythmic agents for atrial and ventricular fibrillation as well as most types of ventricular tachycardia. Lack of ventricular antiarrhythmic efficacy for ICa antagonists at clinically tolerated doses may be because the amount of ICa inhibition is insufficient to prevent or terminate most EADs. Combination of a ICa antagonist with a CaM kinase inhibitor may be effective, however.
The present invention provides methods for treating and preventing arrhythmias in a human subject through the administration of a multifunctional calcium/calmodulin-dependent protein kinase inhibitor to the human in an amount sufficient to suppress early after depolarizations, delayed afterdepolarizations, or intracellular calcium overload.
Also provided are pharmaceutical compositions comprising a multifunctional calcium/calmodulin-dependent protein kinase inhibitor and a pharmaceutically acceptable excipient.
Another aspect of the invention provides for methods of identifying agents useful in the treatment of arrhythmias and provides feasible strategies for the development of a therapeutic, antiarrhythmic drug which can be applied systemically.
In another embodiment, the present invention provides for, methods of treating and preventing arrhythmias in which a multifunctional calcium/calmodulin-dependent protein kinase inhibitor is administered in combination with a second antiarrhythmic agent in order to increase safety and efficacy of the treatment.
In another embodiment, the present invention provides for methods of treating and preventing arrhythmias in which the administration of a multifunctional calcium/calmodulin-dependent protein kinase inhibitoris is combined with treatment of the patient with an antiarrhythmic device.
The present invention also provides for the administration of a multifunctional calcium/calmodulin-dependent protein kinase inhibitor in combination with a second antiarrhythmic agent to block the proarrhythmic effects due to action potential prolongation caused by the second antiarrhythmic agent.